Regulation of Pituitary Hormones. The synthesis and release of prolactin by the pituitary is under complex regulatory control of both the hypothalamus and the gonads. At the molecular level, cis-acting DNA regulatory elements responsible for control of the prolactin gene have been identified by standard DNA sequence analyses. However, nothing is known about the trans-acting factors required for gene activity and the mechanisms of their interactions with the prolactin promoter elements. To address these questions, this laboratory was the first to develop a cell- free in vitro transcription system to study both the cis and trans- acting elements necessary for prolactin expression. Experiments showed GH3, but not HeLa or rat hepatoma, nuclear extracts formed stable DNA-protein complex capable of mediating accurate in vitro transcription of rat prolactin RNA. Deletion mutagenesis demonstrated that only 420 bp of 5' flanking prolactin DNA was necessary for accurate prolactin-specific in vitro transcription. DNA protection experiments showed GH3 trans-acting factors bind to putative prolactin promoter elements. How does the interaction of trans-acting factors with prolactin cis promoter elements activate the stable transcription complex and what elements are responsible for the pituitary-specific gene activity? A series of deletion and single point prolactin mutant templates will be constructed and tested functionally by GH3, HeLa and rat hepatoma in vitro and in vivo transcription assays to identify important cis elements. Mobility shift and DNA protection assays will elucidate nucleotide requirements for affective DNA-protein interactions. To address the issue of prolactin transcription factors, the GH3 nuclear extract proteins will be separated by standard chromatographic techniques. Using the separated prolactin transcription factors combined with the general transcription factors from HeLa cells, the prolactin- specific trans-acting factor(s) will be identified and enriched for. The order of association of the trans-acting factors resulting in a prolactin preinitiation complex will be determined by sequential combinatory preincubations of the separated factors with different templates. This biochemical and genetic dissection of the factors and events leading to an activated in vitro transcription complex will elucidate the basic mechanism of tissue specific prolactin gene expression and provide the essential base for a future understanding of how hormones modulate the system.